Abstract

PURPOSE:

We have investigated the role of t-DARPP in trastuzumab resistance in ERBB2-amplified and overexpressed breast cancer cell lines.

EXPERIMENTAL DESIGN:

We have used the HR-5 and HR-6 trastuzumab-resistant cells that were established from tumors that recurred in the presence of trastuzumab therapy following xenografts of BT-474 cells in nude mice. In addition, SKBR-3 cells, engineered for stable expression of t-DARPP, and HCC-1569 cells, which have constitutive expression of t-DARPP and are de novo resistant to trastuzumab, were used.

RESULTS:

We reported > or =15-fold up-regulation of mRNA and protein levels of t-DARPP in HR-5 and HR-6 cells compared with their progenitor BT-474 trastuzumab-sensitive cells. The t-DARPP expression was not regulated by changes in its promoter DNA methylation levels. The SKBR-3 cells stably expressing t-DARPP developed resistance to trastuzumab compared with their parental cells and empty vector controls (P < 0.01). The trastuzumab-resistant cell lines showed a significant increase in pAKT (Ser(473)) and BCL2 protein levels. The small interfering RNA knockdown of t-DARPP in all trastuzumab-resistant cells led to a significant reduction in ERBB2, pAKT (Ser(473)), and BCL2 protein levels with a significant decrease in cell viability (P < or = 0.001) and an increase in cleaved caspase-3 levels, indicating the progression of these cells toward apoptosis. The t-DARPP protein was associated with both heat shock protein 90 and ERBB2 forming a potential protein complex. This association may play a role in regulating ERBB2 protein in trastuzumab-resistant cells.

CONCLUSION:

We conclude that t-DARPP is a novel molecular target that can mediate the therapeutic resistance to trastuzumab in breast cancer cells.

t-DARPP overexpression correlates with trastuzumab resistance and increased cell survival. Cells were treated with vehicle or trastuzumab (5 and 10 μg/mL) for 24 and 48 h and then subjected to Cell Titer-Glo Luminescent CellViabilityAssay (Promega). A, cell survival was significantly lower in the parental BT-474 cell line compared with trastuzumab-resistant HR-5 and HR-6 cells for all tested trastuzumab concentrations and time points. B, protein extracts from nontreated cells (BT-474, HR-5, and HR-6) were subjected to Western blot analysis of ERBB2 and t-DARPP. ERBB2 protein levels were comparable in all cells. However, t-DARPP protein levels were dramatically higher in HR-5 and HR-6 compared with BT-474 cells. C, similar to trastuzumab-resistant HR-5 and HR-6 cells (A), cell survival of SKBR-3 cells stably expressing t-DARPP (clones 1and 7) was also markedly higher than control SKBR-3 cells following treatment with trastuzumab. D, protein extracts from nontreated SKBR-3-t-DARPP-1, SKBR-3-t-DARPP-7, and control SKBR-3-pcDNA3 cells were subjected to immunoblotting of ERBB2 and t-DARPP. As expected, t-DARPP protein levels were significantly higher in SKBR-3-t-DARPP-1 and SKBR-3-t-DARPP-7 clones compared with SKBR-3-pcDNA3 control cell line. ERBB2 protein levels were comparable in all tested cells. Gel loading was normalized for equal β-actin.

Transcriptional up-regulation of t-DARPP in trastuzumab-resistant cell lines is not associated with gene amplification or promoter hypomethylation. Gene-specific primers for DNA- and mRNA-specific sequences of t-DARPP, ERBB2, β-actin, and HPRT1 were used for PCR and real-time PCR in BT-474, HR-5, and HR-6 cells; the results were normalized to β-actin and HPRT1. A, t-DARPP mRNA expression levels in HR-5 and HR-6 cells were 25- to 100-fold higher compared with BT-474 cells (*, P ≤ 0.001). In contrast, t-DARPP gene amplification levels were similar in all cells. B, ERBB2 mRNA expression and gene amplification levels remained unchanged in all cells. C, DNA bisulfite treatment and pyrosequencing analysis of DNA methylation. DNA from BT-474, HR-5, and HR-6 cells was extracted and modified by bisulfite treatment as described in Materials and Methods. A CpG island from -1,438 to -830 of t-DARPP was amplified with PCR using specific primers. The PCR products were then subjected to pyrosequencing analysis to determine DNA methylation of 10 CpG sites within -1,161 to -1,109 region of t-DARPP. Comparable levels of DNA methylation were detected in all three cell lines, indicating that DNA methylation of t-DARPP CpG island does not regulate its expression in these cell lines.

Knockdown of t-DARPP induces apoptosis in trastuzumab-resistant cells. Terminal deoxynucleotidyl transferase – mediated dUTP nick end labeling assay was done on the trastuzumab-resistant HR-6 cells with scrambled siRNA or t-DARPP siRNA oligonucleotides following thetreatment with 20 μg/mL trastuzumab or vehicle (PBS) for 24 h. The cells were counterstained with 4′,6-diamidino-2-phenylindole (blue fluorescence) and terminal deoxynucleotidyl transferase – mediated dUTP nick end labeling – positive apoptotic cells were visualized with TRITC filter (red fluorescence). Two hundred cells from 20 random fields at ×40(>400cells) were counted. The percentage of apoptosis was determined relative to the total 4′,6-diamidino-2-phenylindole –positive cells. As shown, the results show resistance of HR-6 cells to trastuzumab and >8-fold increase in apoptosis following the knockdown of t-DARPP (P≤0.001).

t-DARPP activates the phosphatidylinositol 3-kinase/AKTsurvival pathway in trastuzumab-resistant cells. A, protein extracts from BT-474, HR-5, and HR-6 cells were analyzed by Western blot for t-DARPP, pAKT (Ser473), AKT, BCL2, and HSP90. B, proteins from HR-5 and HR-6 cells transfected with control scrambled siRNA or t-DARPP siRNA oligonucleotides were also subjected to Western blot analysis of ERBB2 in addition to the proteins described above. pAKT and BCL2 protein levels were markedly higher in HR-5 and HR-6 compared with BT-474 control cells. HSP90 protein levels remained unchanged. Knockdown of t-DARPP in HR-5 and HR-6 cells dramatically decreased ERBB2 and BCL2 protein levels and pAKT but did not affect HSP90 levels. C, protein extracts from SKBR-3-pCDNA3 (vec-con), SKBR-3-t-DARPP-1, and SKBR-3-t-DARPP-7 cells were subjected to Western blot analysis of t-DARPP, AKT, pAKT (Ser473), BCL2, and HSP90. D, protein extracts from SKBR-3-t-DARPP-1 and SKBR-3-t-DARPP-7 cells transfected with control scrambled siRNA or t-DARPP siRNA oligonucleotides were also analyzed by Western blot for ERBB2 and the other proteins described above. As in A, pAKT and BCL2 protein levels were significantly higher in SKBR-3-t-DARPP-1 and SKBR-3-t-DARPP-7 than control cells. Moreover, HSP90 protein levels remained equal in all cells. Knockdown of t-DARPP in SKBR-3-t-DARPP-1 and SKBR-3-t-DARPP-7 cells markedly decreased pAKT, BCL2, and ERBB2 protein levels compared with control cells. Gel loading was normalized for equal β-actin. E, protein extracts from HCC-1569 cells, de novo resistant to trastuzumab, transfected with scrambled siRNA or t-DARPP siRNA oligonucleotides were also analyzed by Western blot for t-DARPP, ERBB2, HSP90, AKT, pAKT (Ser473), BCL2, and caspase-3. As shown in B and D, knockdown of t-DARPP significantly decreased the ERBB2, pAKT (Ser473), and BCL2 protein levels. In addition, HSP90 remained unchanged and caspase-3 was activated.

t-DARPP associates with ERBB2 and HSP90 proteins in trastuzumab-resistant cells. Immunoprecipitated proteins with t-DARPP, HSP90, or ERBB2 antibodies and the cell lysate (Input) from HR-5 cells were resolved on 10% SDS-PAGE and transferred onto Hybond-P polyvinylidene difluoride membranes for Western blot analysis of t-DARPP, HSP90, and ERBB2 proteins. Protein supernatants collected after the first spin of agarose beads were used as controls. Both HSP90 and ERBB2 coprecipitated with t-DARPP.